Structural, biophysical and chemical characterization of redox-sensitive enzymes from the land plant arabidopsis thaliana: alcohol dehydrogenase and glyceraldehyde-3 phosphate dehydrogenase

Redox regulation is a fundamental component of cellular metabolism, controlling the balance between oxidation and reduction reactions that sustain energy production, signalling, and stress responses. Structural biology, particularly X-ray crystallography, provides direct insight into redox-dependent conformational changes and cofactor interactions at the molecular level. The first part of this work focuses on alcohol dehydrogenases (ADH1 and ADH2) from Arabidopsis thaliana and their role in maintaining the NAD⁺/NADH equilibrium during hypoxia and fermentation. ADH1 regenerates NAD⁺ by converting acetaldehyde to ethanol. Biochemical analyses revealed strong pH dependence, with maximal stability under neutral to acidic conditions typical of fermentation. Although ADH1 and ADH2 bind NADH similarly, ADH1 shows weaker NAD⁺ affinity, especially at acidic pH, consistent with its physiological function. The crystal structure of apo-ADH1 revealed an unexpectedly open catalytic conformation, indicating high flexibility of the cofactor-binding domain. Structural comparisons suggested dynamic coupling between the zinc loop and the NAD-binding domain. Structures obtained for both ADH1 and ADH2 with the NADHX inhibitor reveal differences in cofactor binding behaviors. The structure of NADPH bound to ADH1 revealed poor affinity for this cofactor form. Nonetheless both NADH and NADPH protect against aggregation, whereas oxidized forms promote it, suggesting the possibility of a redox sensitive allosteric site at the dimer interface. Four new crystallographic structures were determined, providing insight into cofactor dynamics and protein stability. The second part investigates protection mechanisms against oxidation in cytosolic GAPDH (GAPC1). Two mutants (R231Q and C153S) were generated to study glutathione interactions. While maintaining overall structure, both showed altered phosphate binding at catalytic sites, linking redox sensitivity to structural coordination. Additionally, the glutathione degradation product Cys-Gly lacking the γ-glutamate moiety displayed stronger protective and reactivating effects than GSH, likely due to improved access to the catalytic cysteine. Two additional crystal structures further clarified active-site interactions and redox protection mechanisms